(+)-JQ1 SGCBD01 Selective chemical probe for BET Bromodomains

This probe is available from Cayman Chemical [(+) and (-)], Sigma [(+) and (-)] and Tocris [(+) and (-)]
Racemic mixtures are available from Sigma (+/-) and BPSBioscience (+/-). The concentration of the active ingredient, (+)-JQ1 should be readjusted.

overview
properties
selectivity profile
cell based assay data
references
co-crystal structures

overview

Probe		Negative control

(+)-JQ1 / (+)-SGCBD01		(-)-JQ1 / (-)-SGCBD01

Co-crystal structure

Co-crystal structure of (+)-JQ1/(+)-SGCBD01 in complex with the first bromodomain of BRD4. The inhibitor is shown in ball and stick representation and as transparent cpk demonstrating good shape complementarity to the BRD4 acetyl lysine binding site.

Click on the 'Properties' tab above for more details

Potency Against Target Family

Bromodomain	K _d/nM (ITC)	IC ₅₀/nM (alpha screen)	Tm shift °C
BRD2 (N)	128±6.5	17.7 ± 0.7	6.5 ± 0.1
BRD2 (C)	NT	NT	8.0 ± 0.01
BRD3 (N)	59.5 ± 3.1	NT	8.3 ±0.1
BRD3 (C)	82.0 ±5.3	NT	8.4 ± 0.01
BRD4 (N)	49.0 ± 2.4	76.9 ± 1.7	9.4 ± 0.07
BRD4 (C)	90.1 ± 4.6	32.6 ± 1.8	7.4 ± 0.1
BRDT (N)	190.1 ± 7.6	NT	3.9 ± 0.1
BRDT (C)	NT	NT	NT
CREBBP	ND	12942 ± 640	1.0 ± 0.1

(nt=not tested, nd=not detected)

Shown are binding constants determined by isothermal titration calorimetry (ITC), IC50s determined by a KAc displacement assay (alpha screen) and temperature shift (Tm) data.

Selectivity

Tm shift vs 37 bromodomains all <1°C except BET subfamily, CREBBP (1.2°C) and WDR9 (1.8°C)

Selectivity Beyond Target Family

The racemic mixture was found to be inactive vs 55 receptors and ion channels (CEREP panel, including benzodiazepine GABA _A receptor) at 1µM, except adenosine A3 (61%) & NK2 (56%). Inactive vs 6 lysine methyl transferases up to 100 µM.

Cellular Activity

Fluorescence recovery after photobleaching (FRAP): The panel on the left shows a GFP-BRD4 fluorescent nucleus. The arrow indicates the zone of bleaching. GFP-BRD4 showed significantly quicker recovery in the bleached zone when treated with 500nM JQ1 (right panel).

In vitro Activity

Isothermal titration calorimetry (ITC): The upper panel shows raw injection heats for blank titration of BRD4 into buffer (A), into inactive (-)-JQ1/(-)-SGCBD01 (B) and active (+)-JQ1/(+)-SGCBD01 (C). Normalized binding isotherms are shown in the lower panel for (-)-JQ1/(-)-SGCBD01 (squares) and (+)-JQ1/(+)-SGCBD01 (spheres).

Biology of the BET bromodomains

Bromodomains are protein interaction modules that selectively recognize e-N-acetylated lysine residues (Kac). This recognition process is the molecular basis of the "reading process" of epigenetic acetylation marks. In the human proteome there are 41 diverse proteins containing 57 bromodomains which share a conserved ford of left-handed helical bundles. This arrangement creates a deep largely hydrophobic acetyl lysine binding cavity which constitutes an attractive pocket for the development of selective protein interaction inhibitors. The human BET family (BRD2, BRD3, BRD4 and BRDT) which all contain two conserved bromodomains per target, plays a key role regulating transcription of growth stimulating genes. In collaboration with the laboratory of James E. Bradner we developed a potent, cell permeable and selective inhibitor for BET bromodomains ((+)-JQ1) and evaluated its role in NUT midline carcinoma, an aggressive incurable cancer that is genetically define by a chromosomal translocation of BRD4 with NUT (Nuclear protein in testis). Details of this study have been published in Nature (doi:10.1038/nature09504).

properties

[(S)-4-(4-Chloro-phenyl)-2,3,9-trimethyl-6H-1-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-acetic acid tert-butyl ester
Click here to download SDF file

Physical and chemical properties
Molecular weight	456.1
Molecular formula	C₂₃H₂₅ClN₄O₂S
IUPAC name	[(S)-4-(4-Chloro-phenyl)-2,3,9-trimethyl-6H-1-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6-yl]-acetic acid tert-butyl este
logP	4.0
PSA	53.7 A
No. of chiral centres	1
No. of rotatable bonds	5
No. of hydrogen bond acceptors	6
No. of hydrogen bond donors	0
Storage	Stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended
Dissolution	Soluble in DMSO at least up to 10mM

SMILES:
CC1=NN=C2N1C3=C(C(C4=CC=C(C=C4)Cl)=N[C@H]2CC(OC(C)(C)C)=O)C(C)=C(S3)C
InChI:
InChI=1S/C23H25ClN4O2S/c1-12-13(2)31-22-19(12)20(15-7-9-16(24)10-8-15)25-17(11-18(29)30-23(4,5)6)21-27-26-14(3)28(21)22/h7-10,17H,11H2,1-6H3/t17-/m0/s1
InChIKey:
DNVXATUJJDPFDM-KRWDZBQOSA-N

selectivity profile

Selectivity Within Target Family

DSF Assay	(-)JQ1		(+)JQ1/SGCBD01
Protein	ΔTm	STD	ΔTm	STD
ASH1L	0.00	0.00	0.00	0.00
ATAD2	0.31	0.23	0.18	0.68
BAZ2B	0.00	0.00	0.00	0.00
BRD1	0.59	0.73	0.00	0.00
BRD2/1	0.89	0.60	6.47	0.09
BRD2/2	0.91	0.06	7.97	0.01
BRD3/1	1.95	0.23	8.27	0.11
BRD3/2	2.14	0.47	8.39	0.01
BRD4/1	1.12	0.09	9.35	0.07
BRD4/2	0.21	0.11	7.44	0.14
BRD9	0.00	0.00	0.00	0.00
BRDT/1	0.38	0.02	3.93	0.13
BRPF1	0.76	0.18	0.00	0.00
CECR2	0.00	0.00	0.00	0.00
CREBBP	1.18	0.21	1.04	0.11
EP300	0.46	0.22	0.07	0.21
FALZ	0.00	0.00	0.00	0.00
GCN5L2	0.00	0.00	0.00	0.00
KIAA1240	0.14	0.22	0.05	0.28
LOC93349	0.00	0.00	0.00	0.00
PB1/1	0.00	0.00	0.00	0.00
PB1/2	0.48	0.11	0.00	0.00
PB1/3	0.98	0.01	0.79	0.05
PB1/5	0.37	0.25	0.00	0.00
PB1/6	0.00	0.00	0.00	0.00
PCAF	0.00	0.00	0.00	0.00
PHIP/2	0.00	0.00	0.00	0.00
SMARCA2	0.95	0.04	0.93	0.02
SMARCA4	0.60	0.18	0.15	0.10
SP140	0.69	0.80	0.00	0.00
TAF1/2	0.56	0.04	0.28	0.05
TAF1/3	0.33	0.03	0.50	0.04
TAF1L/2	0.00	0.00	0.04	0.04
TAF1L/3	0.14	0.18	0.00	0.00
TIF1	0.00	0.00	0.00	0.00
TRIM28/4,5	0.00	0.00	0.00	0.00
WDR9	0.00	0.00	0.00	0.00

Isothermal titration calorimetry (ITC)

Protein	[P] (µM)	[L] (µM)	K_d (nM)	ΔH^obs (kcal/mol)	N	TΔS (kcal/mol)	ΔG (kcal/mol)
BRD2(1)	225	25	128.4 ± 6.5	-7.74 ± 0.003	1.02 ± 0.002	1.35	-9.08
BRD3(1)	306	25	59.5 ± 3.1	-6.57 ± 0.017	1.07 ± 0.002	2.97	-9.54
BRD3(2)	280	30	82.0 ± 5.3	-4.93 ± 0.017	1.03 ± 0.002	4.41	-9.35
BRD4(1)	240	25	49.0 ± 2.4	-8.42 ± 0.019	1.00 ± 0.001	1.22	-9.64
BRD4(2)	250	27	90.1 ± 4.6	-3.22 ± 0.009	1.06 ± 0.002	5.76	-9.29
BRDT(1)	305	25	190.1 ± 7.6	-8.30 ± 0.024	1.06 ± 0.002	0.56	-8.86
CREBBP	950	30	nd	nd	nd	nd	nd
WDR9(2)	300	25	nd	nd	nd	nd	nd

Nd: not detected

Selectivity Beyond Target Family

CEREP Assay

Displacement of a tetra-acetylated histone H4 peptide by JQ1/SGCBD01 isomers using BRD4(1), BRD4(2) or of an acetylated H3 peptide using CREBBP.

Materials and Methods

Differential Scanning Fluorimetry (DSF)

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 µM in 20 µl volume. Compounds were added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence during the protein denaturation process was approximated by the equation

where ΔuG_(T) is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant and y_F and y_U are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein respectively. The baselines of the denatured and native states were approximated by a linear fit. The observed temperature shifts, ΔT_m^obs, were recorded as the difference between the transition midpoints of sample and reference wells containing protein without ligand in the same plate and determined by non-linear least squares fit.

Isothermal Titration Calorimetry

Experiments were carried out on a VP-ITC titration microcalorimeter from MicroCal^TM, LLC (Northampton, MA). All experiments were carried out at 15 °C while stirring at 295 rpm, in ITC buffer (50 mM HEPES pH 7.4 at 25 °C, 150 mM NaCl). The injection syringe (250 µl) was loaded with a solution of the protein sample (300 µM protein for the BETs, 950 µM protein for CREBBP and 600 µM for WDR9(2), in ITC buffer). All titrations were conducted using an initial injection of 2 µl followed by 34 identical injections of 8 µl with a duration of 16 sec (per injection) and a spacing of 250 sec between injections. The heat of dilution was determined by independent titrations (protein into buffer) and was subtracted from the experimental data. The collected data were implicated in the MicroCal^TM Origin software supplied with the instrument to yield enthalpies of binding (ΔH) and binding constants (K_B) as previously described by Wiseman and coworkers⁵⁰. Thermodynamic parameters were calculated (ΔG = ΔH - TΔS = -RTlnK_B, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed.

CEREP assay

JQ1/SGCBD01 (1 µm) was screened against a panel of 55 ligand receptors, ion channels and transports using an established and widely utilized commercial assay platform (ExpresSProfile; CEREP, Paris, FRANCE).

Alpha screen Assay

All reagents were diluted in 50 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 supplemented with 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150–0 µM and 4 µl transferred to low-volume 384-well plates (ProxiPlateTM-384 Plus, PerkinElmer, USA), followed by 4 µl of HIS-tagged protein (BRD4(1), 250 nM, BRD4(2) and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 µl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH; peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 µl of streptavidin-coated donor beads (25 µg/ml) and 4 µl nickel chelate acceptor beads (25 µg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set. IC50 values were calculated in Prism 5 after normalization against corresponding DMSO controls and are given as the final concentration of compound in the 20 µl reaction volume.

in vitro potency

cell based assay data

FRAP Assay

Fluorescence recovery after photobleaching (FRAP) of GFP–BRD4 demonstrates enhanced recovery in the presence of JQ1/SGCBD01. Nuclei are false-coloured in proportion to fluorescence intensity. White circles indicate target regions of photobleaching (left panel). The kinetics of the fluorescence recovery of JQ1/SGCBD01 treated cells (red) and control (black) is shown in the panel in the middle and recovery rates are shown at the right panel. Data represent the mean ± s.d. (n = 5), and are annotated with P-values as obtained from a two-tailed t-test.

Immunohistochemistry

JQ1/SGCBD01 prompts squamous differentiation, growth arrest and apoptosis in vivo, as determined by IHC. Histopathological analysis of NMC 797 tumors excised from mice treated with JQ1 (right panel) reveals squamous differentiation (H&E), effacement of nuclear NUT foci (NUT), impaired proliferation (Ki67) and induction of keratin expression all as compared to vehicle-treated animals. Animals were treated once a day (50mg/kg, IP) using the racemic mixture of JQ1/SGCBD01

Materials and Methods

Fluorescence Recovery After Photobleaching (FRAP)

FRAP studies were performed on U2OS cells transfected (lipofectamine; Invitrogen) with mammalian overexpression constructs encoding GFP chimera with BRD4, NUT and BRD4-NUT. A 5 µm2 nuclear region was bleached with high laser intensity in one cell within each field, and measured for recovery with low laser intensity and a 150 µm pinhole. Images of identical fields were acquired using a Nikon C1 Plus confocal microscope equipped with a 37 °C heated chamber and FRAP modules. Average intensities of the bleached region were measured over time and using MetaMorph v7, and normalized to an independent region of interest before bleaching. Data were then analyzed to assess the time to half-maximal fluorescence recovery and the mobile fraction in Microsoft Excel Mac 12.2.4.

Immunohistochemistry

Immunohistochemistry was performed using the Aperio Digital Pathology Environment (Aperio Technologies, Vista, CA) at the DF/HCC Core Laboratory at the Brigham and Women’s Hospital. Slides were scanned in an automated fashion on a ScanScope XT Instrument at 20x resolution with the ScanScope Console v10 (Aperio Technologies). Digital images were remotely analyzed using ImageScope (Aperio Technologies). IHC images were exported as high-resolution TIFF files with comparable settings for paired data.

references

Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature (24 September 2010) doi:10.1038/nature09504 Letter (http://www.nature.com/nature/journal/vnfv/ncurrent/abs/nature09504.html)
Ptashne, M. Binding reactions: epigenetic switches, signal transduction and cancer. Curr. Biol. 19, R234–R241 (2009).
Schreiber, S. L. & Bernstein, B. E. Signaling network model of chromatin. Cell 111, 771–778 (2002).
Marushige, K. Activation of chromatin by acetylation of histone side chains. Proc. Natl Acad. Sci. USA 73, 3937–3941 (1976).
Owen,D. J. et al.The structural basis for the recognition of acetylated histoneH4 by the bromodomain of histone acetyltransferase gcn5p. EMBO J. 19, 6141–6149 (2000).
Yang, Z. et al. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol. Cell 19, 535–545 (2005).
Rahl, P. B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).
Yang, Z., He, N. & Zhou, Q. Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression. Mol. Cell. Biol. 28, 967–976 (2008).
French, C. A. et al. BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19). Am. J. Pathol. 159, 1987–1992 (2001).
Dey,A. et al.A bromodomain protein,MCAP, associates withmitotic chromosomes and affects G2-to-M transition. Mol. Cell. Biol. 20, 6537–6549 (2000).
You, J. et al. Regulation of aurora B expression by the bromodomain protein Brd4. Mol. Cell. Biol. 29, 5094–5103 (2009).
Abbate, E. A., Voitenleitner, C. & Botchan, M. R. Structure of the papillomavirus DNA-tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association. Mol. Cell 24, 877–889 (2006).
Huang, B., Yang, X. D., Zhou, M. M., Ozato, K. & Chen, L. F. Brd4 coactivates transcriptional activation ofNF-kBvia specific binding to acetylated RelA.Mol. Cell. Biol. 29, 1375–1387 (2009).
Matzuk MM, McKeown MR, Filippakopoulos P, Li Q, Ma L, Agno JE, Lemieux ME, Picaud S, Yu RN, Qi J, Knapp S, Bradner JE. Small-molecule inhibition of BRDT for male contraception. Cell 150(4):673-84 (2012).

pk properties

co-crystal structures

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Main features

synthetic schemes

materials and methods

UNC0638 Selective chemical probe for G9a/GLP methyltransferases

This probe is available from Cayman Chemical, Sigma (hydrate) and Tocris.

overview
properties
selectivity profile
cell based assay data
co-crystal structures

overview

Probe		Negative control

UNC0638		UNC0737

Biology of the G9a/GLP methyltransferases

G9a (EHMT2) and GLP (EHMT1) catalyze the mono and dimethylation of lysine 9 of histone 3 (H3K9) and other non-histone substrates such as p53 and WIZ. UNC0737, the N-methyl analog of UNC0638, was a poor inhibitor of G9a and GLP. The combination of the high structural similarity between UNC0737 and UNC0638 and the >300-fold loss of potency in UNC0737 compared to UNC0638 makes UNC0737 an appropriate negative control for use in cellular and functional assays.

Cellular Activity

Significant reduction in H3K9 dimethylation at 100nM in MDA-MB231 cells as measured by fluorescence immunostaining without significant cellular toxicity.

Click on the 'Cell-based Assay Data' tab above for more details

Selectivity Within Target Family

Protein	IC₅₀/nM (Activity)	Tm shift °C ¹
G9a (EHMT2)	<15 (hill slope 1.3)	4
GLP (EHMT1)	19±1 (hill hlope 0.8)	8
SETD7	>10,000	nt
SETD8	>10,000	nd
PRMT3	>10,000	nd
SUV39H2	>10,000	nt
DOT1L	nt	nd
PRDM1	nt	nd
PRDM10	nt	nd
PRDM12	nt	nd
SMYD3	nt	nd
JMJD2E	4660 (AlphaScreen)	nt
HTATIP	nt	nd

(nt=not tested, nd=not detected, ¹ singlicate determination @ 100 µM)

Selectivity Beyond Target Family

>30% Inhib @ 1µM

Receptor	%Inhib
Adrenergic alpha_1A	90
Adrenergic alpha_1B	69
Muscarinic M₂	64

Click on the 'Selectivity Profile' tab above for more details

properties

2-cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy) quinazolin-4-amine
Click here to download SDF file

Physical and chemical properties
Molecular weight	509.7
Molecular formula	C₃₀H₄₇N₅O₂
IUPAC name	2-cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy) quinazolin-4-amine
logP	5.78
PSA	62.0 A²
No. of chiral centres	0
No. of rotatable bonds	10
No. of hydrogen bond acceptors	6
No. of hydrogen bond donors	1
Storage	Stable as solid in the dark at -20°C.
Dissolution	Soluble in DMSO at least up to 10mM

SMILES:
CC(C)N1CCC(NC2=NC(C3CCCCC3)=NC4=C2C=C(OC)C(OCCCN5CCCC5)=C4)CC1
InChI:
InChI=1S/C30H47N5O2/c1-22(2)35-17-12-24(13-18-35)31-30-25-20-27(36-3)28(37-19-9-16-34-14-7-8-15-34) 21-26(25)32-29(33-30)23-10-5-4-6-11-23/h20-24H,4-19H2,1-3H3,(H,31,32,33)
InChIKey:
QOECJCJVIMVJGX-UHFFFAOYSA-N

selectivity profile

Selectivity Within Target Family

Protein	IC₅₀/nM (Activity)	Tm shift °C ¹
G9a (EHMT2)	<15	4
GLP (EHMT1)	19 ± 1	8
SETD7	>10,000	nt
SETD8	>10,000	nd
PRMT3	>10,000	nd
SUV39H2	>10,000	nt
DOT1L	nt	nd
PRDM1	nt	nd
PRDM10	nt	nd
PRDM12	nt	nd
SMYD3	nt	nd
JMJD2E	4660 (AlphaScreen)	nt
HTATIP	nt	nd

(nt=not tested, nd=not detected, ¹ singlicate determination @ 100 µM)

Target	IC₅₀ / nM (Activity)
DNMT1	1287**
MLL	>10,000**
EZH2	>10,000**
PRMT1	>10,000**
SUV39H1	>10,000**
SUV39H2	>10,000**
G9a	91**

**Screened at BPS Bioscience using different format

		Tm shift °C
Protein	Screening Methods	UNC0638 µM
Protein	Screening Methods	1	10	100	500
DOT1L	DSF
PRDM1	DSF
PRDM10	DSF
PRDM12	DSF
SMYD3	DSF
HIATIP	DSF
EHMT1	DSF		5	8	6
G9a	DSF		2	4	4
SETD8	DSLS
PRMT3	DSLS

Blank box indicates Tm shift of <2 °C

Selectivity Beyond Target Family

Radioligand binding performed at Ricerca

>30% Inhib @ 1 µM

Receptor	%Inhib
Adrenergic alpha_1A	90
Adrenergic alpha_1B	69
Muscarinic M₂	64

<30% Inhib @ 1 µM

Receptor
Transporter, Norepinephrine (NET)	Glutamate, NMDA, Phencyclidine
Nicotinic Acetylcholine Alpha1, Bungarotoxin	Adenosine A_2A
Dopamine D_2S	Calcium Channel L-Type, Dihydropyridine
GABAA, Flunitrazepam, Central	GABAA, Muscimol, Central
Sodium Channel, Site 2	Histamine H₁
Potassium Channel hERG	Adenosine A₁
Cannabinoid CB₁	Rolipram
Dopamine D₁	Potassium Channel[KATP]
Nicotinic Acetylcholine	Adrenergic beta₁
Adrenergic beta₂	Prostanoid EP₄
Muscarinic M₃	Serotonin (5-Hydroxytryptamine) 5-HT2B
Opiate mu (OP3, MOP)	Phorbol Ester
Adrenergic alpha_2A	Sigma1

Peptide Displacement Measured by FP (click image for larger version)

Materials and Methods

Activity Assay

Histone methyltransferase assay was performed using a coupled assay originally developed by Collazo et al. 2005. In this assay SAHH (S-adenosylhomocysteine hydrolase) and adenosine deaminase convert the methyltransferase reaction product (S-adenosylhomocysteine) to homocysteine and inosine. Homocysteine can be quantified using Thioglo-1 (Calbiochem). Substrate peptides used in this assay were: the first 25 residues of histone 3 [H3 (1-25)] for G9a, EHMT1 and SETD7 at 10, 20 and 100 µM respectively; the first 24 residues of histone 4 [H4 (1-24)] at 10 and 500 µM for PRMT3 and SETD8 respectively; and H3K9Me1 [H3 (1-15), monomethylated at lysine 9] at 200 µM for SUV39H2. The assay mixtures were prepared in 25 mM potassium phosphate buffer pH 7.5, 1 mM EDTA, 2 mM MgCl₂, 0.01% Triton X-100 with 5 µM SAHH , 0.3 U/ml of adenosine deaminase from Sigma, 25 µM SAM, and 15 µM Thioglo-1. G9a (25 nM), EHMT1 (100 nM), SUV39H2 (100 nM), SETD7 (200 nM) and PRMT3 (1 µM) were assayed in the presence of UNC0638 at concentrations ranging from 4 nM to 16 µM. After 2 min incubation, reactions were initiated by the addition of above mentioned histone peptides. The methylation reactions were followed by monitoring the increase in fluorescence using BioTek Synergy2 plate reader with 360/40 nm excitation filter and 528/20 nm emission filter for 20 min in 384-well format. SETD8 (250 nM) was assayed under the same conditions; however the Thioglo-1 was added at the end of the reaction for quantification. The peptide and protein background were subtracted. IC50 values were calculated using Sigmaplot and the standard deviations were calculated from two independent experiments. SAHH clone was provided by Dr. Trievel, University of Michigan.

Differential Scanning Fluorimetry (DSF)

DOT1L, PRDM1, PRDM10, PRDM12, SMYD3, HIATIP, G9a, EHMT1 and SETD7 were screened for binding to UNC0638 by DSF. A real-time PCR device (RTPCR 480 II) from Roche was used to monitor protein unfolding by monitoring the increase in the fluorescence of the fluorophor SYPRO Orange (Invitrogen, Carlsbad, CA) as described before (Niesen et al. 2007; Vedadi et al. 2006). Protein samples ranging from 0.05 to 0.2 mg/mL in 100 mM Hepes buffer (pH 7.5) containing 150 mM NaCl, and 0, 1, 10 and 100 µM of the compound were screened. Compound dilutions were made from stock solutions of 100% DMSO. The final concentration of DMSO was kept at 0.2% throughout the dilutions. All these solutions contained 5x Sypro Orange. 20 µL aliquots were transferred to a 384-well PCR plate and scanned at a heating rate of 1 °C/min from 20 to 95 °C. Fluorescence intensities were plotted as a function of temperature by using an internally developed software package (Vedadi et al. 2006).

Differential Static Light Scattering (DSLS)

SETD8 and PRMT3 were screened for binding to UNC0638 by DSLS. Temperature-dependent aggregation was measured by using static light scattering (StarGazer) (Vedadi et al. 2006, Senisterra et al. 2006). Fifty microliters of protein (0.4 mg/ml) was heated from 27°C to 85°C at a rate of 1°C per min in each well of a clear-bottom 384-well plate (Nunc, Rochester, NY) in the presence of 0, 1, 10 and 100 µM of UNC0638. Incident light was shone on the protein drop from beneath at an angle of 30°. Protein aggregation was monitored by measuring the intensity of the scattered light every 30 s with a CCD camera. The pixel intensities in a preselected region of each well were integrated to generate a value representative of the total amount of scattered light in that region. These total intensities were then plotted against temperature for each sample well and fitted to the Boltzman equation by nonlinear regression. The resulting point of inflection of each resulting curve was defined as the Tagg.

Peptide Displacement

Fluorescence polarization (FP) measurements were performed in 384 well-plates, using Synergy 4 microplate reader from BioTek. H3 (1-15) peptide (ARTKQTARKSTGGKA) was synthesized, N-terminally labeled with fluorescein [F-H3 (1-15)] and purified by Tufts University Core Services (Boston, MA, USA). Displacement of F-H3 (1-15) peptide was monitored using the fluorescence polarization signal obtained upon peptide binding to G9a protein. G9a (4 µM in 20 mM Tris pH 8.0, 250 mM NaCl, 500 µM SAH, and 0.01% Triton) was incubated with 40 nM F-H3 (1-15) peptide, and different concentrations of BIX-01294 (purchased from Sigma-Aldrich), UNC0224, UNC0638 and unlabeled H3 (1-25) peptide from 0.1 to 100 µM were added. Displacement of peptide was monitored by following the decrease in FP signal. Data were normalized and plotted as percentage, and fit to a hyperbolic function using Sigma Plot software.

in vitro potency

cell based assay data

Cellular Activity
(BIX-01294 reported by Kubicek et al, 2007 Molecular Cell 25, 473-481)

H3K9Me2 immunostaining and cell viability measured in MDA-MB231 cells after 48h exposure to BIX-01294 or UNC0638. UNC0638 exhibits a dose-dependant reduction in H3K9 dimethylation, while being more potent and efficacious than BIX-01294.

Cellular Toxicity

UNC0638 clearly shows an improved toxicity profile both in absolute terms and relative to its cell-based activity. This property will enable the use of UNC0638 without concern about possible complications of cellular toxicity.

	In vitro G9a IC₅₀(nM)	H3K9Me2 48h IC₅₀(nM)	Tox 48h EC₅₀(nM)	Tox/Func Ratio
BIX-01294	133 ± 15	500 ± 43	2805	5.6
UNC0638	<15	81 ± 9	11190	138

Poor separation of functional and toxic effects

Good separation of functional and toxic effects

Materials and Methods

MDA-MB231 cells were cultured in RPMI with 10% FBS and MCF7 cells cultured in DMEM with 10% FBS.

MTT Toxicity Assay

Cells were grown in the presence or absence of inhibitors for stated amount of time. The media was removed and replaced with DMEM 10% FBS without phenol red supplemented with 1mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and incubated for 1-2h. Live cells reduce yellow MTT to purple formazan. The resulting formazan was solubilized in acidified isopropanol and 1% Triton and absorbance measured at 570nm, corrected for 650nm background.

In-Cell Western (ICW)

Cells were grown in 96-well plates in the presence of inhibitors as stated in figures. Media was removed by flicking and 2% formaldehyde in PBS added for 15min. After five washes with 0.1% Triton X100 in PBS, cells were blocked for 1h with 1% BSA in PBS. Three out of four replicates were exposed to primary H3K9m2 antibody, Abcam #1220 at 1/800 dilution in 1% BSA, PBS for 2h. One replicate was reserved for background control. The wells were washed five times with 0.1% Tween 20 in PBS, then secondary IR800 conjugated antibody (LiCor) and DNA-intercalating dye, DRAQ5 (LiCor) added for 1h. After 5 washes with 0.1% Tween 20 in PBS, the plates were read on Odyssey (LiCor) scanner at 800nm (H3K9m2 signal; 764nm excitation) and 700nm (DRAQ5 signal; 683nm excitation). Fluorescence intensity was quantified, normalized to background and DRAQ5 signal expressed as percentage of control.

Gene knockdown

shRNAs were obtained from The RNAi Consortium (TRC) (Dr J Moffat) and processed and used as outlined in the TRC protocols.
G9a shRNA: NM_025256.4-3163s1c1; EHMT1 shRNA NM_024757.3-346s1c1; Control promegaLuc_221s1c1

references

pk properties

co-crystal structures

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Human euchromatic histone-lysine N-methyltransferase 2 (G9a/EHMT2) in complex with UNC0638 (pdb code 3NNI)
3RJW: Dong A, Wasney GA, Tempel W, Liu F, Barsyte D, Allali-Hassani A, Chen X, Chau I, Hajian T, Senisterra G, Chavda N, Arora K, Siarheyeva A, Kireev DB, Herold JM, Bochkarev A, Bountra C, Weigelt J, Edwards AM, Frye SV, Arrowsmith CH, Brown PJ, Jin J, Vedadi M
PDB Code: 3RJW (deposited on 15.Apr.11)

Datapack version: 1 (built on 28.Jun.11; last revised on 29.Jun.11)

Crystal structure of the methyltransferase domain of human G9a/EHMT2 in complex with S-adenosyl homocysteine (SAH) and UNC0638
UNC0638 bound to the G9a/EHMT2. [pocket view] [residues view]
UNC0638 occupies the substrate lysine and neighbouring arginine binding sites
[Inhibitor vs peptide]
[Overview]

Note: The target annotations and structure descriptions within this datapack are compiled by our Principal Investigators and are not peer-reviewed. If you find anything in the annotations that is not accurate, please notify us using the our on-line feedback page or send an e-mail to isee@sgc.ox.ac.uk.

synthetic schemes

materials and methods

02.03.2007

Unique public-private research partnership leads world in determining 3D structure of proteins related to human disease

by: SGC

Structural Genomics Consortium reaches project milestone ahead of schedule

2 March 2007, Toronto – The Structural Genomics Consortium (SGC), an international research effort set up in 2004 to determine the three-dimensional structures of proteins relevant to human disease, today posted its 375th protein structure in the public domain. The SGC, which is the largest international research project ever directed from Canada, operates from laboratories at University of Toronto, University of Oxford, and Karolinska Institutet, Stockholm.

25.05.2005

Structural Genomics Consortium Celebrates First Anniversary with 50 New Protein Structures Ahead of Plan

by: SGC

Oxford, UK and Toronto, Canada, May 25, 2005 -The Structural Genomics Consortium (SGC; www.thesgc.com), an Anglo-Canadian charitable consortium of public and private agencies, today announces it has delivered its first 50 human and malaria protein structures into the public domain on budget and 2 months ahead of schedule. New data that this provides to research will expedite the development of new and improved medicines and provide tools for researchers to study important diseases.

03.04.2003

UK-Canadian consortium commits $95-m to international health research project

by: SGC

April 3, 2003 - Unravelling the structure of hundreds of human proteins will be the goal of an ambitious $95 million partnership that brings together British and Canadian health researchers under the direction of an internationally renowned Canadian scientist.

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(+)-JQ1 SGCBD01 Selective chemical probe for BET Bromodomains

Co-crystal structure

Potency Against Target Family

Selectivity

Selectivity Beyond Target Family

Cellular Activity

In vitro Activity

Biology of the BET bromodomains

Selectivity Within Target Family

Selectivity Beyond Target Family

Materials and Methods

Differential Scanning Fluorimetry (DSF)

Isothermal Titration Calorimetry

CEREP assay

Alpha screen Assay

FRAP Assay

Immunohistochemistry

Materials and Methods

Fluorescence Recovery After Photobleaching (FRAP)

Immunohistochemistry

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UNC0638 Selective chemical probe for G9a/GLP methyltransferases

Biology of the G9a/GLP methyltransferases

Cellular Activity

Selectivity Within Target Family

Selectivity Beyond Target Family

Selectivity Within Target Family

Selectivity Beyond Target Family

Materials and Methods

Activity Assay

Differential Scanning Fluorimetry (DSF)

Differential Static Light Scattering (DSLS)

Peptide Displacement

Cellular Activity (BIX-01294 reported by Kubicek et al, 2007 Molecular Cell 25, 473-481)

Cellular Toxicity

Materials and Methods

MTT Toxicity Assay

In-Cell Western (ICW)

Gene knockdown

Please wait whilst the interactive viewer is loaded!

Unique public-private research partnership leads world in determining 3D structure of proteins related to human disease

Structural Genomics Consortium reaches project milestone ahead of schedule

Structural Genomics Consortium Celebrates First Anniversary with 50 New Protein Structures Ahead of Plan

UK-Canadian consortium commits $95-m to international health research project

Cellular Activity
(BIX-01294 reported by Kubicek et al, 2007 Molecular Cell 25, 473-481)