Researchers from Oxford University, GlaxoSmithKline and Cellzome, as well as Memorial Sloan Kettering Hospital have developed and characterised the first selective inhibitor for a specific class of histone demethylases and demonstrated its utility in inhibiting proinflammatory cytokine production in human macrophages.
CARLSBAD, Calif., July19, 2012 – Life Technologies Corporation (NASDAQ: LIFE) today announced it has signed a collaborative partnership with the Structural Genomics Consortium (SGC), and several groups of leading expert scientists in antibody technology at the Universities of Chicago and Toronto, to generate a first-ever master set of quality epigenetic recombinant antibodies for use in disease-related research. The first 58 of 200 highly-specific antibodies to be designed are now available to the scientific community.
The probe GSK-J1 is available from Tocris, Cayman Chemical (sodiumsalt) and Sigma .
The less-active control GSK-J2 is available from Tocris, Cayman Chemical (sodiumsalt) and Sigma (sodiumsalt).
The pro-drug GSK-J4 is available from Tocris, Cayman Chemical (hydrochloride) and Sigma.
The pro-drug GSK-J5 is available from Tocris (hydrochloride), Cayman Chemical (hydrochloride) and Sigma.
| Probe | Prodrug of GSK-J1 for cellular use | Less-active control | Prodrug of GSK-J2 for cellular use | |||
 |  |  |  | |||
GSK-J1 | GSK-J4 | GSK-J2 | GSK-J5 |
Co-crystal structure of JMJD3 in complex with GSKJ1 (PDB 4ASK). Residues within in the catalytic pocket of JMJD3 are shown as silver sticks, GSK-J1 is shown in orange sticks and Co2+ , that replaces the endogenous Fe2+ , is shown as a yellow sphere. (Click to enlarge)
Details of this study have been published in Nature - A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response.
Methylated lysine residues of histone 3 lysine 27 (H3K27me) play important roles in regulating gene activity by silencing through the polycomb-repressive complex (PRC1 or PRC2). This epigenetic mark can be demethylated by lysine demethylase (KDM) enzymes UTX and JmjD3 in humans and play important roles in cellular differentiation, development and cancer.
Here we present in collaboration with GlaxoSmithKline the first selective and potent histone demethylase inhibitor (GSK-J1) that has significant activity (IC50 60 nM for human JmjD3) in vitro and in cells using an ester derivative (GSK-J4: 1 µM < IC50 < 10 µM; e.g. 9 µM in primary human macrophages). The pyridine regio-isomer GSK-J2 displays significantly less on-target activity (IC50 > 100 µM for human JmjD3) and thus can be used as control for target effects in vitro, and as ester derivative (GSK-J5) in cells. Recent data has shown that GSK-J1 also shows some activity (IC50 950 nM for Jarid1b, IC50 1.76 uM for Jarid1c) against H3K4me3/2/1 demethylases.
 |
| 3-{[2-(pyridin-2-yl)-6-(2,3,4,5-tetrahydro-1H-3-benzazepin-3-yl)pyrimidin-4-yl]amino}propanoic acid Click here to download SDF file |
| Physical and chemical properties | |
|---|---|
| Molecular weight | 389.46 |
| Molecular formula | C22H23N5O2 |
| IUPAC name | 3-{[2-(pyridin-2-yl)-6-(2,3,4,5-tetrahydro-1H-3-benzazepin-3-yl)pyrimidin-4-yl]amino}propanoic acid |
| clogP | 2.23 |
| PSA | 91.24Å2 |
Compounds GSK-J1 and GSK-J2 were screened against a range of Jmj enzymes using the specified assays. GSK-J1 also does not significantly inhibit 100 protein kinases at a concentration of 30 µM in a competition binding assay and had negligible off-target activity against a panel of 60 unrelated proteins, including other chromatin-modifying enzymes such as histone deacetylases (see also Supplementary Table 4 and 5, data taken from primary reference (Kruidenier et al., Nature 2012)).
Profiling of GSK-J1 in a Tm shift assay (differential scanning fluorimetry) against a panel of human 2’OG oxygenases (see phylogenetic profile) reveals significant Tm shifts with H3K27me3 demethylases (> 2.5 C) but not with any other member, suggesting no significant crossreactivities for this H3K27 demethylase inhibitor. Known histone lysine demethylase subfamilies (KDM) are indicated. Recent alphascreen data has shown that GSK-J1 can also inhibit the H3K4me3/2/1 demethylases JARID1B/C (see table).
| Selectivity | ||||||
|---|---|---|---|---|---|---|
| Alpha Screen | GSK-J1 Mean IC50 (µM) | GSK-J2 Mean IC50 (µM) | 2-OG concentration (µM) | Fe2+ Final Concentration (µM) | Peptide substrate and concentration (µM) | AlphaScreen Detection Antibody |
| Human JmjD3 | 0.06 | >100 | 10 | 10 | Biotin-H3(14-34)K27Me3 0.06 µM | Millipore 07-452 (0.4 mg/ml) |
| Human JmjD2a | 44.3 | - | 10 | 1 | Biotin-H3(1-15)K9Me3 0.03 µM | Abcam ab1220 (0.05 mg/ml) |
| Human JmjD2e | 19.6 | - | 10 | 1 | Biotin-H3(1-15)K9Me3 0.03 µM | Abcam ab1220 (0.05 mg/ml) |
| Human JmjD1a | 41.0 | - | 5 | 10 | H3(1-21)K9Me2-Biotin 0.06 µM | Abcam ab8896 (0.4 mg/ml) |
| Jarid1b | 0.95 | 5 | 10 | H3(1-21)K4Me3-Biotin 0.1 µM | Cell Signaling Technology 9726S (1:3000) | |
| Jarid1c | 1.76 | 5 | 10 | H3(1-21)K4Me3-Biotin 0.1 µM | Cell Signaling Technology 9726S (1:3000) | |
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.
The effects of GSK-J4 (ethyl ester derivative of GSK-J1) were assessed in LPS stimulated human macrophages derived from monocytes or PBMC- in this system the compound shows an inhibitory effect on pro-inflammatory cytokine (e.g. TNF production) with an IC 50 of 9 µM. No significant adverse effects were observed at 20 µM in proliferating human primary fibroblasts.
| Assay | System | IC50 |
|---|---|---|
| Inhibition of TNF release | Primary human macrophages derived from PBMC (healthy volunteers or rheumatoid arthritis patients) | 9 µM |
| Increase in global H3K27me3 levels (immunofluorescence) | HeLa cells | < 10 µM (estimated) |
A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Laurens Kruidenier, Chun-wa Chung, Zhongjun Cheng, John Liddle, KaHing Che, Gerard Joberty, Marcus Bantscheff, Chas Bountra, Angela Bridges, Hawa Diallo, Dirk Eberhard, Sue Hutchinson, Emma Jones, Roy Katso, Melanie Leveridge, Palwinder K. Mander, Julie Mosley, Cesar Ramirez-Molina, Paul Rowland, Christopher J. Schofield, Robert J. Sheppard, Julia E. Smith, Catherine Swales, Robert Tanner, Pamela Thomas, Anthony Tumber, Gerard Drewes, Udo Oppermann, Dinshaw J. Patel, Kevin Lee & David M. Wilson.
Nature, 2012, 488, 404–408
Antibodies are first released in massive collaboration to create ‘master set’ of high-quality renewable epigenetics antibodies
This probe is available from Tocris, Sigma and Cayman Chemical.
This probe (as trifluoroacetate salt) is available from Cayman Chemical.
The control may be requested by clicking here.
| Probe |
 |
SGC0946 |
Protein methyltransferases (PMTs) play essential roles in epigenetic regulation of gene expression and chromatin dependent signaling via their methylation of histones and other chromatin associated substrates. Mutation or misregulation of PMTs is linked to many diseases, especially cancer, and there is strong interest in this family of proteins as potential drug targets. Most PKMTs contain a conserved SET-domain, with the exception of DOT1L which has a protein fold that more closely resembles PRMTs. DOT1L is also unique in that it is the only PKMT to methylate histone H3 on Lysine 79 (H3K79), a chromatin mark associated with active chromatin and transcriptional elongation. Here we present SGC0946, a potent and selective inhibitor of DOT1L, which potently and selectively kills cells containing an MLL translocation. SGC0946 inhibits DOT1L with an IC50 of 0.3nM in a radioactive enzyme assay and is over 100-fold selective for other HMTs. In addition, SGC0946 potently reduces H3K79 dimethylation with cellular IC50 of 2.6nM in A431 cells, and 8.8nM in MCF10A cells.
|
1-(3-((((2R,3S,4R,5R)-5-(4-amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea |
| Physical and chemical properties | |
|---|---|
| Molecular weight | 653.2 |
| Molecular formula | C28H40BrN7O4.HCl |
| IUPAC name | 1-(3-((((2R,3S,4R,5R)-5-(4-amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea |
| logP | 3.51 |
| PSA | 120.4 A |
| Storage | Stable as solid in the dark at -20°C. |
SGC0946 selectively inhibits DOT1L
Inhibition of H3K79me2 by a series of DOT1L inhibitors in A431 cells (4 day treatment).
Catalytic site remodeling of the DOT1L methyltransferase by selective inhibitors
Wenyu Yu, Emma J. Chory, Amy K. Wernimont, Wolfram Tempel, Alex Scopton, Alexander Federation, Jason J. Marineau, Jun Qi, Dalia Barsyte-Lovejoy, Joanna Yi, Richard Marcellus, Roxana E. Iacob, John R. Engen, Erno Wienholds, Fengling Li, Javier Pineda, Guillermina Estiu, Tatiana Shatseva, Taraneh Hajian, Rima Al-awar, John E. Dick, Masoud Vedadi, Peter J. Brown, Cheryl H. Arrowsmith *, James E. Bradner *, Matthieu Schapira *. Nature Communications 3 : 1288doi:10.1038/ncomms2304 (2012)
Main features
This probe is available from Cayman Chemical, Sigma and Tocris
| Probe |
 |
IOX2 |
Biology of the hypoxia inducible factor (HIF) prolyl-hydroxylases
Levels of the Hypoxia Inducible Factor (HIF), a master regulator of the cellular-response to hypoxia, are regulated by the post-translational modification of prolyl-residues in oxygen-dependent degradation domains in the HIFα subunit. HIFα prolyl-hydroxylation signals for its degradation by the proteasome. The requirement of the HIF prolyl-hydroxylases (PHD or EGLN enzymes) for dioxygen as a co-substrate enables them to act as the hypoxia-sensing component of the HIF system. PHDs are members of the 2-oxoglutarate (2OG-dependent) oxygenase superfamily. Inhibition of the PHDs reduces HIFα prolyl-hydroxylation thereby elevating HIF levels and artificially inducing the hypoxic response. Because HIF-target genes include those encoding for biomedically important proteins such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF), PHD inhibition is of considerable medical interest.
IOX2: A chemical probe for HIF prolyl-hydroxylases
IOX2, a chemical probe for HIF prolyl-hydroxylases is the result of collaborations with the University of Sevilla, Genome Institute of Singapore, University of Sheffield, University of Oxford, BioNanotechnology Research Center KRIBB and the SGC [1].
Potency Against Target Family
IOX2 displayed an AlphaScreen IC50 of 22nM for inhibition of PHD2
Selectivity Beyond Target Family
IOX2 was found to be inactive against a panel of 55 receptors and ion channels (CEREP panel) at 10µM.
Co-crystal structure
The co-crystal structure of IOX2 with PHD2 has NOT been solved, however, there is a crystal structure with a close analogue (pdb id: 4BQW). This analogue has a methyl rather than benzyl group on the isoquinoline nitrogen. Please click on the 'Co-Crystal structures' tab above for more details.
Cellular Activity
IOX2 showed selective inhibition (by Western blot) of PHD catalysed HIF-1α prolyl-hydroxylation in a variety of human cell lines including renal carcinoma (RCC4) cell line lacking VHL, embryonic kidney (293T), bone osteosarcoma (U2OS) and RCC4 reexpressing VHL (RCC4/VHL) cells.
 |
(1-benzyl-4-hydroxy-2-oxo-1, 2-dihydroquinoline-3-carbonyl)glycine |
| Physical and chemical properties | |
|---|---|
| Molecular weight | 352.35 |
| Molecular formula | C19H16N2O5 |
| IUPAC name | (1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbonyl)glycine |
| logP | -3.99 |
| PSA | |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 5 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 3 |
| Storage | Store at -20°C |
| Dissolution | Soluble to 100 uM in DMSO |
An inactive, negative control for IOX2 has not yet been identified
IOX2 displayed an AlphaScreen IC50 of 22nM for inhibition of PHD2; values for each data point are averages ± standard deviation (n≥3).
IOX2 was profiled against a panel of human histone Nε-methyl lysine demethylase 2OG-oxygenases in an AlphaScreen assay to determine selectivity.
IOX2 was found to be inactive against a panel of 55 receptors and ion channels (CEREP panel) at 10µM.
Engagement of IOX2 in an in vitro cell assay
Efficacy of IOX2 in a human renal carcinoma (RCC4) cell line lacking VHL (required for the degradation of hydroxylated HIF-α) was established, using antibodies selective for hydroxylated form(s) of a peptide corresponding to the HIF-1α C-terminal oxygen-dependent degradation domain to probe for PHD catalysed inhibition of HIF prolyl-hydroxylation in comparison to FIH catalysed asparaginyl-hydroxylation. IOX2 was further tested in VHL-competent cell lines, including human embryonic kidney (293T), human bone osteosarcoma (U2OS) and RCC4 reexpressing VHL (RCC4/VHL) cells, where increased HIF-1α levels were used as an indicator for PHD inhibition. IOX2 effectively increased HIF-1α levels in all tested VHL-competent cell lines showing that the inhibitory effects are independent of cell type. These results establish that IOX2 is a potent and selective cellular PHD inhibitor.
Selective inhibition of IOX2 in human cell lines. Selectivity of the tested inhibitors for HIF-1α prolyl- over asparaginyl- hydroxylation in RCC4 after 6h of treatment. Upregulation of HIF-1α by inhibitors in various cell types (after 6h of treatment): HEK293T, U2OS and RCC4 stably-transfected with C-HA-tagged wildtype VHL.
A co-crystal structure of PHD2’s catalytic domain with a methyl group on the isoquinoline nitrogen, compound 1, (rather than benzyl group in IOX2) group has been solved (pdb id 4BQW).
Compound 1
The bicyclic heteroaromatic ring of IOX2 is sandwiched between the hydrophobic side chains of Tyr310, Met299 and Trp389. IOX2 coordinates Mn(II) (substituting for Fe(II)) in a bidentate fashion with the side chain amide-carbonyl and phenolic oxygen. The side chain carboxylate of IOX2 is positioned to hydrogen bond with Arg383 and Tyr329. The Tyr303 phenol is positioned to hydrogen bond to the isoquinoline-2-OH. The aromatic rings of IOX2 project through the active site opening and likely make a steric clash with the hydroxylated HIF-1α.
This tool compound is available from Cayman Chemical, Sigma and Tocris.
| Probe |
 |
IOX1 |
Biology of the 2-oxoglutarate and ferrous iron dependent oxygenases
Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nε-Methyl lysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription.
2-oxoglutarate (2OG) oxygenases are a ubiquitous and biologically important family of enzymes [1]. In human their roles include catalysing steps in collagen and carnitine biosynthesis, lipid metabolism, nucleic acid repair and modification, hypoxic sensing as well as histone demethylation. Impaired 2OG oxygenase activity is linked to the cellular hypoxic response and various diseases including cancer. Non-selective generic inhibitors of 2OG oxygenases include iron-chelators, Co(II) ions and 2OG analogues. The most widely used 2OG analogues are pyridine-2, 4-dicarboxylate and N-oxalylglycine. However, these compounds suffer from the lack of breadth of selectivity and their poor cell permeability often necessitates the use of pro-drug diester derivatives
IOX1: A tool compound for 2-oxoglutarate oxygenases
IOX1 a broad-spectrum, cell permeable inhibitor of most 2OG oxygenases which does not require application in a pro-drug form [2]. It was discovered following a high throughput screening campaign against the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases [3]. A cell permeable n-octyl ester analogue of IOX1 with an improved cellular potency has recently been reported [4].
Potency Against Target Family
The co-crystal structures of IOX1 with JMJD2A (pdb id: 3NJY), JMJD3, (pdb id: 2XXZ) and FIH, (pdb id: 3OD4) have been solved. OX1 inhibits by binding to the active site Fe(II). Click on the 'Co-Crystal structures' tab above for more details.
Selectivity Beyond Target Family
IOX1 was found to be inactive against a panel of 55 receptors and ion channels (CEREP panel) at 10µM.
Cellular Activity
IOX1 exhibited cellular potency in a HeLa cell immunofluorescence assay, with EC50 values of 86 uM (KDM4A), 37 uM (KDM6B) and 24 uM (KDM2A).
IOX1
 |
8-hydroxyquinoline-5-carboxylic acid |
| Physical and chemical properties | |
|---|---|
| Molecular weight | 189.17 |
| Molecular formula | C10H7N1O3 |
| IUPAC name | 8-hydroxyquinoline-5-carboxylic acid |
| logDpH7.4 | -1.55 |
| PSA | 69.9 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 1 |
| No. of hydrogen bond acceptors | 4 |
| No. of hydrogen bond donors | 2 |
| Storage | Store at 4°C |
| Solubility | Soluble to 100 mM in DMSO and to 100 mM in 1eq. NaOH |
An inactive, negative control compound for IOX-1 has not yet been identified
SMILES:
OC1=CC=C(C(O)=O)C2=CC=CN=C21
InChI:
InChI=1S/C10H7NO3/c12-8-4-3-7(10(13)14)6-2-1-5-11-9(6)8/h1-5,12H,(H,13,14)
InChIKey:
JGRPKOGHYBAVMW-UHFFFAOYSA-N
Potency against Target (JmjC-domain containing histone lysine demethylases)
IOX1 was screened against 10 JmjC-domain containing histone lysine demethylases by AlphaScreen or by MALDI-TOF Mass Spectroscopy. IOX1 showed nanomolar affinity for all except KDM2A and PHF8
Binding of the IOX1 may also indirectly affect substrate binding, supported by the observation of mixed mode inhibition kinetics when IOX1 was tested with varying concentrations of 2OG in a formaldehyde dehydrogenase assay.
Selectivity
IOX1 was screened against 4 other active 2OG Oxygenases where it showed moderate to weak activity
Selectivity Beyond Target Family
IOX1 was found to be inactive against a panel of 55 receptors and ion channels (CEREP panel) at 10µM.
Engagement of IOX1 in a cell assay
PHD2 and FIH are both inhibited in cells as demonstrated by lowered HIF1α hydroxylation levels (hydroxylated-Pro564 (PHD2) and hydroxylated-Asn803 (FIH)).
Using an indirect immunofluorescence assay to directly measure demethylase activity in vivo by prevention of depletion of H3K9me3 in JMJD2A overexpressing HeLa cells. Indirect immunofluorescence with anti-Flag (green), anti-H3K9me3 (red), and DAPI staining (blue) in HeLa cells overexpressing Flag-tagged JMJD2A. DMSO solvent treatment has no effect on JMJD2A demethylase activity (white arrows) while increasing concentrations of IOX1 treatment (100 µM to 300µM range) resulted in gradual increases in H3K9Me3 levels. The JMJD2A H188A enzymatic mutant does not affect H3K9Me3 levels when overexpressed.
Treatment with increasing IOX1 concentrations showed a dose-dependent increase in H3K9me3 fluorescence intensity, demonstrating that H3K9me3 demethylation by JMJD2A is inhibited by IOX1 in cells. The cellular IC50 value for IOX1 was determined to be 86.5 µM. Notably, this compound does not require modification to a “pro-drug” ester form in order to be active in cells, unlike previously reported N-oxalylglycine derivatives [5].
A similar effect was noted in JMJD3A overexpressing cells this time measuring the effect on H3K27me3
X-ray crystal structures of IOX1 in complex with various 2OG oxygenases; JMJD2A (pdb id: 3NJY), JMJD3, (pdb id: 2XXZ) and FIH, (pdb id: 3OD4) and AlkB (pdb id: 4JHT) have been solved.
King et al. 2010 PLoSOne 5(11)e15535
Crystallisation of IOX1 in complex with JMJD2A revealed that IOX1 is positioned in a similar location to the 2OG analogue N-oxalylglycine in complex with JMJD2A and Ni(II) (which substitutes for Fe(II)); the 5-carboxy group of IOX1 and the C-5 carboxylates are positioned to interact with side chains Lys 206, Tyr 132. IOX1 is also positioned to coordinate with the active site Ni(II), in a bidentate fashion via its quinoline-nitrogen and 8-hydroxy group. Comparison of this structure with that of JMJD2A in complex with the H3K9me3 substrate peptide and N-oxalylglycine (PDB ID: 2OQ6) shows that the binding of the IOX1 is likely to compete with binding of 2OG and not directly with the peptide substrate.
Alpha Screen Assay
All reagents were diluted in 50 mM HEPES, 0.1 % BSA, pH 7.5 supplemented with 0.01 % Tween20 and allowed to equilibrate to room temperature prior to addition to plates. Catalytic turnover assays were run in 10µL volumes in 384-well plates at RT with enzyme (0.5-25nM), biotinylated substrate peptide (30-1000nM), Fe(II) (1-10µM), Ascorbate (100µM), 2OG (5-40µM). EDTA was used to quench the reaction (5µL) and AlphaScreen donor (Streptavidin-conjugated) and acceptor (ProteinA-conjugated) beads preincubated with peptide product antibodies were added (5µL). Plates were protected from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech) using an AlphaScreen 680nm excitation/570nm emission filter set. IC50 values were calculated after normalization against corresponding DMSO controls.
Matrix-Assisted Laser Desorption/Ionisation-Time-of-flight (MALDI-TOF) Mass Spectrometry
KDM assays were carried out as reported [3] using an assay reaction consisting of JMJD2 (1 µM), Ferrous ammonium sulphate (10 µM), Ascorbate (100 µM), 2OG (10 µM), Histone H3 peptides (10 µM) in 50 mM HEPES (pH 7.5) with varying concentrations of inhibitors. The reaction was incubated at RT and 1:1 quenching with methanol followed by addition of four volumes of 20 mM triammonium citrate.
Cell-Based Assays
Flag-tagged JMJD2A was transiently overexpressed in HeLa cells either in the presence of a vehicle control (DMSO), 1 mM dimethyl-2,4-PDCA (a cell-permeable derivative of 2,4-PDCA), 2.5 mM dimethyloxalylglycine (DMOG, a cell-permeable derivative of N-oxalylglycine), or varying concentrations of IOX1. After 24 hours of incubation time cells were fixed and then analyzed by indirect immunofluorescence with Flag tag antibody to identify the cells overexpressing the demethylase and an antibody recognizing endogenous H3K9me3 to quantify the level of this histone modification. A series of images were collected for each treatment on a standard epifluorescence microscope and CellProfiler was used to analyze the images for DAPI signal thereby identifying the location of individual cells and create a boundary that delineates the volume of the nuclear compartment. As not all cells in a given field are transfected, the Flag-JMJD2A-expressing cells were identified by quantifying the immunofluorescence signal resulting from the Flag tag antibody staining and using the mock transfected cells as a baseline for the signal intensity of non-transfected cells. Once the transfected cells were identified, the nuclear H3K9me3 immunofluorescence signal for each cell was quantified by CellProfiler. The levels of H3K9me3 staining intensity were analyzed in the DMSO vehicle treated or inhibitor treated samples. As a control and a means of determining maximal possible inhibition of demethylase activity, cells expressing the JMJD2A H188A catalytically deficient mutant were also quantified in each experiment. The level of demethylase activity inhibition by IOX1 treatment was determined by quantifying the immunofluorescence signal from the DMSO treated sample (100% demethylase activity) compared to the maximal theoretical inhibition signal intensity as determined by the H3K9me3 signal in cells expressing the catalytically deficient JMJD2A H188A mutant (0% demethylase activity). For each treatment a minimum of 400 transfected cells was analyzed and the final values of inhibition were derived from inhibition experiments carried out on three separate days.
This probe is available from Cayman Chemical, Sigma and Tocris
| Probe |
 |
PFI-1 |
Details of this chemical probe have been published in the Journal of Medicinal Chemistry - Identification of a chemical probe for BET bromodomain inhibition through optimization of a fragment-derived hit
Biology of the BET bromodomains
Bromodomains are protein interaction modules that selectively recognize ε-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. PFI-1 has been developed in collaboration with Pfizer.
Tocris is offering PFI-1 through their catalogue: (Cat. no. 4445)
| Potency | |
|---|---|
| In vitro | |
| BET family member | IC50 µM |
| BRD2 2nd Bromodomain (Alphascreen) | 0.098 |
| BRD4 1st Bromodomain (Alphascreen) | 0.22 |
| BRD2 1st Bromodomain (Octet Red) | 0.123 |
| BRD4 1st Bromodomain (ITC) | 0.136 |
| BRD4 2nd Bromodomain (ITC) | 0.303 |
| In cells | |
| BRD4 (FRAP assay) | Accelerated FRAP recovery at 1 uM |
 |
| 2-Methoxy-N-(3-methyl-2-oxo-1,2,3,4-tetrahydro-quinazolin-6-yl)-bezenesulfonamide Click here to download SDF file |
| Physical and chemical properties | |
|---|---|
| Molecular weight | 347.1 |
| Molecular formula | C16H17N3O4S |
| IUPAC name | 2-methoxy-N-(3-methyl-2-oxo-1,2,3,4-tetrahydroquinazolin-6-yl)benzenesulfonamide |
| logP | 1.3 |
| PSA | 96 |
| Storage | -20 as powder. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | Soluble in DMSO at least up to 10mM |
InChI=1S/C16H17N3O4S/c1-19-10-11-9-12(7-8-13(11)17-16(19)20)18-24(21,22)15-6-4-3-5-14(15)23-2/h3-9,18H,10H2,1-2H3,(H,17,20)
InChIKey:
TXZPMHLMPKIUGK-UHFFFAOYSA-N
Selectivity screening data of PFI-1 using temperature shift assays. Screened targets are highlighted in bold. Temperature shifts are indicated by red filled circles with increasing radii for higher Tm values as indicated in the figure
| Selectivity | |
|---|---|
| Bromodomains | |
| Target | Tm shift °C @ 10 µM |
| BRD2A 1st Bromodomain | 4.6 |
| BRD2 2nd Bromodomain | 5.3 |
| BRD3 1st Bromodomain | 5.2 |
| BRD3 2nd Bromodomain | 5.5 |
| BRD4 1st Bromodomain | 6.5 |
| BRD4 2nd Bromodomain | 3.8 |
| BRDT 1st Bromodomain | 2.1 |
| CREBBP | 2.7 |
| BAZ2B | 0.3 |
| LOC93349 | 0.1 |
| PB1~5 | 0.8 |
| PCAF | 0.8 |
| Other proteins | |
| Invitrogen 50 kinase panel | <20% inhibition @ 1 µM |
BRD2 KD=0.123µM
Off Target CREBBP KD=49.5µM
> 350 fold selective against BRDs outside BET
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. PFI-1 was 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 ?u G (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, ? T 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) 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, 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. Heats of dilution were determined by independent titrations (protein into buffer) and were subtracted from the experimental data. Data analysis was carried out using the MicroCal TM Origin software supplied with the instrument to yield enthalpies of binding (?H) and binding constants (K B). Thermodynamic parameters were calculated (? G = ? H - T? S = -R TlnK B, where ? G, ? H and ? S are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed.
CEREP assay
PFi-1 (10 µm) was screened against a panel of 15 ligand receptors, ion channels and transports using an established and widely utilized commercial assay platform (ExpresSProfile; CEREP, Paris, FRANCE); <50% inhibition was observed.
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 1500 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), 50 nM, BRD4(2), 50 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 BETs H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-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.
Bio-Layer Interferometry
Kinetic measurements were done using OctetRed384 instrument (ForteBio Inc, CA, USA). Biotinylated protein was immobilized on Super Streptavidin Biosensors at 2µg/ml concentration. Association and dissociation measurements were done in 50 mM HEPES, 100 mM NaCl, pH 7.4 buffer supplemented with 0.01 % Tween. Experiments were performed at 25°C with association and dissociation times of 240 sec. Compounds were prepared as one in two dilutions starting from 128µM. Binding to the reference sensors (no protein attached) was subtracted before calculations. Binding constants were calculated using ForteBio Analysis software.
b
| Potency | |
|---|---|
| In cells | |
| BRD4 (FRAP assay) | Accelerated FRAP recovery at 1 µM |
| LPS challenge assay in PBMCs | EC50 1.89 µM (n=6) for inhibition of IL6 production |
Fluorescence Recovery After Photobleaching (FRAP)
FRAP studies were performed on U2OS cells transfected (Fugene HD; Roche) with mammalian overexpression constructs encoding GFP fused to the N-terminus of full length BRD4. Compounds were added as indicated 16 h post transfection. The FRAP and imaging system consisted of a Zeiss LSM 710 scanhead (Zeiss GmbH, Jena, Germany) coupled to an inverted Zeiss Axio Observer.Z1 microscope equipped with a high-numerical-aperture (N. A. 1.3) 40x oil immersion objective (Zeiss GmbH, Jena, Germany). Samples were placed in an incubator chamber capable of maintaining temperature (37 °C) and humidity. FRAP and GFP fluorescence imaging were carried out with an argon-ion laser (488nm) and with a piezomultiplier tube (PMT) detector set to detect fluorescence between 500-550nm. Once an initial scan had been taken, a region of interest corresponding to approximately 50% of the entire GFP positive nucleus was empirically selected for bleaching. A time lapse series was then taken to record GFP recovery using 1% of the power used for bleaching. The image datasets and fluorescence recovery data were exported from ZEN 2009, the microscope control software, into Microsoft Excel to determine the average half-time for full recovery for 10-20 cells per treatment point. The average intensity at each imaging time was normalized to an independent region of interest before bleaching.
PFI-1 shows considerably accelerated FRAP recovery at 1 µM
Fluorescent Recovery After Photo-bleaching (FRAP) experiments using full-length GFP-BRD4. Nuclei of U2OS cells transfected with GFP-BRD4 were treated with the DMSO control (upper panel), 1 mM or 5 mM PFI-1 (middle and lower panel). Photographs were taken at 0, 10, 20 and 30 s after photo-bleaching of half the nucleus
Half recovery time measured when treating cells with PFI-1 (1 μM, 5 μM) show faster recovery than the DMSO control. (a) Half times of fluorescence recovery of DMSO and PFI-1 (1, 5 μM) treated cells. (b) Time dependence of fluorescent recovery in the bleached area for DMSO (black box and line) and PFI-1 1 μM and 5 μM (red box and line, blue box and line, respectively) treated cells.
Antiproliferative effects of PFI-1 on MLL tumour cells
Differentiation of mll-AF9 leukemic blasts induced by PFI-1. Shown are murine mll-AF9 transformed blast cells in the presence of DMSO (left) and 5 mM PFI-1 after 48h exposure.
Dose-response of the anti-proliferative effects of PFI-1 on leukaemic cell lines
Identification of a chemical probe for BET bromodomain inhibition through optimization of a fragment-derived hit
Paul Fish, Panagis Filippakopoulos, Gerwyn Bish, Paul Brennan, Mark Edward Bunnage, Andrew Cook, Oleg Fedorov, Brian S Gerstenberger, Hannah Jones, Stefan Knapp, Brian Marsden, Karl Nocka, Dafydd R Owen, Martin Philpott, Sarah Picaud, Michael Primiano, Michael Ralph, Nunzio Sciammetta, and John Trzupek
J. Med. Chem., 2012, 55 (22), 9831–9837
PFI-1 - A highly Selective Protein Interaction Inhibitor Targeting BET bromodomains
Sarah Picaud, David Da Costa, Angeliki Thanasopoulou, Panagis Filippakopoulos, Paul V. Fish, Martin Philpott, Oleg Fedorov, Paul Brennan, Mark E. Bunnage, Dafydd R. Owen, James E. Bradner, Philippe Taniere, Brendan O’Sullivan, Susanne Müller-Knapp, Juerg Schwaller, Tatjana Stankovic, Stefan Knapp
Cancer Res, 2013, 73, 3336-3346
(a)
(b)
(a) PFI-1 bound to BRD4 ε-N-acetylated lysine (Kac) binding pocket; (b) PFI-1 forms polar contacts with the conserved asparagine and interacts with many hydrophobic and aromatic residues in the BET Kac binding pocket. PDB ID: 4E96
London, September 28, 2011 - The international Structural Genomics Consortium (SGC) today announced $48.9 million in new funding has been attained. This renewed investment will allow the SGC to further develop its open access research program to support drug discovery and the development of new medicines. The announcement comes as the SGC on June 30 successfully completed its second phase of funding (2007-2011).
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